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(Circulation Research. 2004;94:1367.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Rho Kinase Mediates Cold-Induced Constriction of Cutaneous Arteries

Role of 2C-Adrenoceptor Translocation

S.R. Bailey, A.H. Eid, S. Mitra, S. Flavahan, N.A. Flavahan

From the Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus.

Correspondence to Nicholas A. Flavahan, Heart and Lung Research Institute, R 110E, The Ohio State University, 473 West 12th Ave, Columbus, OH 43210. E-mail flavahan-1{at}medctr.osu.edu

	Abstract

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Cold-induced vasoconstriction in cutaneous blood vessels is mediated in part by increased activity of vascular smooth muscle 2-adrenoceptors (VSM 2-ARs). In mouse cutaneous arteries, 2C-ARs are normally silent at 37°C but mediate cold-induced augmentation of 2-AR responsiveness. In transfected HEK293 cells, this functional rescue is mediated by cold-induced translocation of 2C-ARs from the Golgi to the plasma membrane. Experiments were performed to determine the role of Rho/Rho kinase signaling in this process. Inhibition of Rho kinase (fasudil, Y27632 or H-1152) did not affect constriction of isolated mouse tail arteries to the 2-AR agonist UK 14 304 at 37°C but dramatically reduced the augmented responses to the agonist at 28°C. After Rho kinase inhibition, cooling no longer increased constriction evoked by 2-AR stimulation. Cooling (to 28°C) activated Rho in VSM cells and increased the calcium sensitivity of constriction in toxin-permeabilized arteries. Stimulation of 2-ARs in VSM cells had no effect on Rho activity or calcium sensitivity at 37°C or 28°C. In HEK293 cells transfected with 2C-ARs, cooling (to 28°C) stimulated the translocation of 2C-ARs to the plasma membrane and this effect was prevented by inhibition of Rho kinase, using fasudil or RNA interference. Consistent with inhibition of the spatial rescue of 2C-ARs, fasudil inhibited 2-AR–mediated mobilization of calcium in tail arteries at 28°C but not 37°C. Therefore, cold-induced activation of Rho/Rho kinase can mediate cold-induced constriction in cutaneous arteries by enabling translocation of 2C-ARs to the plasma membrane and by increasing the calcium sensitivity of the contractile process.

Key Words: raynaud phenomenon • siRNA • thermoregulation • HEK293 cells

	Introduction

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Cold-induced vasoconstriction in the cutaneous circulation is a protective physiological response that acts to reduce heat loss. The constriction results from a reflex increase in sympathetic output and a direct local effect of cold to increase the activity of nerve-released norepinephrine.¹ This local effect is mediated by a cold-induced, selective augmentation of 2-adrenoceptor (2-AR) reactivity on vascular smooth muscle cells (VSMs).^2–4 Local cold-sensitivity is increased in patients with Raynaud phenomenon and Scleroderma who exhibit cold-induced peripheral vasospasm, which can be prevented by 2-AR blockade.⁵

2-ARs have been classified by pharmacological and molecular techniques into 2A-AR, 2B-AR, and 2C-AR subtypes.⁶ Thermosensitivity of cutaneous blood vessels is mediated by 2C-ARs.⁷ In cutaneous arteries of the mouse tail, 2-AR constriction at 37°C is mediated by 2A-ARs, with no apparent contribution from 2C-ARs. However, after moderate cooling, 2C-ARs are no longer silent and mediate the remarkable cold-induced augmentation of 2-AR reactivity. After transfection in HEK 293 cells, 2A-ARs are expressed on the cell surface and respond to activation by regulating adenylyl cyclase activity. Moderate cooling does not influence 2A-AR location or function.⁸ In contrast, 2C-ARs are not functional at 37°C and are localized, by subcellular fractionation and immunofluorescent analysis, to the Golgi compartment.⁸ A similar mislocalization of 2C-ARs is also observed in transfected COS-7, NRK, MDCK, and rat1 fibroblast cells.^6,9,10 Moderate cooling stimulates redistribution of 2C-ARs to the cell surface and rescues the 2C-AR functional response, demonstrated by agonist-dependent inhibition of adenylyl cyclase.⁸ The mechanisms contributing to cold-induced translocation of 2C-ARs have so far not been identified.

Rho is a member of the Ras family of small GTP-binding proteins and cycles between a GDP-bound inactive state and a GTP-bound active state.^11–13 Rho plays a central role in regulating actin/myosin-dependent processes in VSMs, including contractility and motility.^13,14 Smooth muscle myosin ATPases are activated by actin after phosphorylation of regulatory myosin light chains (MLC) by a calcium-calmodulin–dependent myosin light chain kinase (MLCK). Conversely, they are inactivated after dephosphorylation of MLC by a calcium-independent MLC phosphatase (MLCP).¹⁴ The Rho effector, Rho kinase,¹⁵ inhibits MLCP, increasing phosphorylation of MLC and causing contraction of VSMs in the absence of an increase in intracellular calcium concentration.¹⁴ This mechanism has been identified in a number of vascular beds including the human internal mammary artery.^14,16 Studies have also implicated Rho in the process of translocation and membrane targeting of transmembrane proteins, such as Na⁺K⁺-ATPase.¹⁷

The aim of the present study was to investigate whether the Rho/Rho kinase signaling pathway plays a role in cold-induced modulation of 2-ARs in cutaneous arteries.

	Methods

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Blood Vessel Preparation
Male mice (C57BL/6) were killed by CO₂ asphyxiation. Small tail arteries (200 to 300 µm internal diameter) were removed and placed in cold Krebs-Ringer bicarbonate solution containing (in mmol/L): 118.3 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 2.5 CaCl₂, 25.0 NaHCO₃, and 11.1 glucose. They were cannulated in a microvascular chamber (Living Systems, Burlington, Vt) and maintained at a constant transmural pressure of 60 mm Hg in the absence of flow.⁷ Internal diameter was monitored as previously described.⁷ Concentration-effect curves to UK 14 304 were determined in the absence and presence of the selective Rho kinase inhibitors, Y27632 (0.3 µmol/L), fasudil (3 µmol/L), or H-1152 (0.1 µmol/L). Arteries were incubated with the inhibitors for 30 minutes before and during the subsequent exposure to the agonist. When analyzing the influence of cold on 2-AR responsiveness, the temperature of the superfusate was decreased to 28°C for 30 minutes before administration of UK 14 304.⁷

Rhotekin Binding Assay for RhoA-GTP
Human cutaneous arteriolar VSMs were grown to 80% confluence in Ham’s growth medium, then quiesced in Ham’s serum-free medium for 72 hours.¹⁸ The medium was then replaced with precooled medium at 28°C or washed with medium at 37°C as control. RhoA activation was assessed using a precipitation assay for RhoA-GTP, according to the manufacturer’s instructions (Upstate Biotechnology, Lake Placid, NY).

Permeabilization Studies
Permeabilization of endothelium-denuded arteries was accomplished using 500 U/mL -toxin¹⁹ in a pCa²⁺ 9.0 intracellular substitution solution for 20 minutes. The substitution solution was adapted from Akopov et al²⁰ and comprised (in mmol/L): Mg acetate,⁶ EGTA,⁵ imidazole,²⁰ DTT,¹ phosphocreatine,¹² Mg ATP,⁵ and leupeptin (0.1) plus calmodulin (0.001) and creatine phosphokinase (15 U/mL). CaCl₂ was added to give the desired free calcium concentration (WinMAXC version 2.40),²¹ with potassium acetate added to give a final ionic strength of 150 mmol/L. The Ca²⁺ diameter relationship was determined by increasing the Ca²⁺ concentration from pCa²⁺ 9.0 to 5.0.

Quantitation of Cell Surface 2C-AR
HEK 293 cells (American Type Culture Collection, Manassas, Va) were transiently transfected with either pCDNA3 expression vector (mock) or pCDNA3-2C-AR with hemagglutinin (HA) tagged to the amino terminal domain of the receptor, as previously described.⁸ Forty-eight hours after transfection, the media was replaced with precooled media at 28°C or washed with media at 37°C and maintained at those temperatures for 60 minutes. The cells were then rinsed (ice-cold phosphate-buffered saline) and incubated with anti-HA monoclonal antibody (1:200 dilution) for 60 minutes at 4°C to label cell surface 2C-ARs. The cells were subsequently rinsed (phosphate-buffered saline, 4°C), then lysed in phosphate-buffered saline containing 1% digitonin, 0.5% deoxycholate, and protease inhibitors for 1 hour at 4°C. Cell lysates were processed and incubated with protein A/G-Sepharose beads for 1 hour at 4°C to precipitate the antibody-bound surface receptors. 2C-ARs were resolved by SDS-PAGE and detected using Western blotting with a rabbit polyclonal antibody directed against the receptor.⁸ When analyzing the effect of the Rho kinase inhibitor, fasudil (10 µmol/L), cells were incubated with the inhibitor for 30 minutes before and during the 1-hour incubation at 37°C or 28°C.

RNA Interference
Four silencing RNA (siRNA) oligoduplexes that target different sequences of Rho kinase (ROCK-I) were obtained from Qiagen (Valencia, Calif).²² The sequences targeted by the siRNA duplexes were (relative to the start codon): duplex 1, 566 to 584; duplex 2, 639 to 657; duplex 3, 1958 to 1976; and duplex 4, 2780 to 2798. A nonsilencing siRNA was used as control (Qiagen). In preliminary experiments, the combination of duplexes 1 and 4 provided the most effective inhibition of ROCK-I expression and was used in subsequent experiments.

HEK293 cells were cultured in 100-mm dishes in MEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, and 1 mmol/L sodium pyruvate, without antibiotics. HEK 293 cells were cotransfected using Lipofectamine2000 (Invitrogen, Carlsbad, Calif) with 4 µg of pCDNA3-2C-AR and a nonsilencing, control RNA duplex (480 pmol) or the combination of siRNA duplexes 1 and 2 (240 pmol each). Live cell labeling of surface 2C-ARs was performed 72 hours after transfection.

Calcium Imaging
Endothelium-denuded arteries were cannulated in a perfusion myograph chamber with an integral glass coverslip for use with a confocal microscope (chamber model CH/1; Living Systems, Burlington, Vt). Arteries were loaded with the calcium probe Fluo-4AM at a concentration of 5 µmol/L in 0.05% dimethylsulfoxide and 0.001% Pluronic acid for 60 minutes at 37°C. Fluo-4AM was then removed and the arteries allowed to recover for 30 minutes before imaging (Zeiss LSM 510 confocal laser scanning microscope). Longitudinal sections through the mid-part of the vessel were obtained to provide an image containing both sides of the vascular wall. Changes in fluorescence intensity were determined using image analysis software (Zeiss LSM 510). Arteries underwent repeated stimulation with UK 14 304 (0.1 µmol/L). An initial response was obtained at 37°C, after which the temperature was either maintained at this level or reduced to 28°C for 30 minutes. When analyzing the effect of the Rho kinase inhibitor fasudil (3 µmol/L) or the 2C-AR antagonist MK912 (0.3 nmol/L), they were present for 30 minutes before and during administration of UK 14 304. The responses of control arteries to UK 14 304 in the absence of any inhibitors were also recorded to confirm that the magnitude of the response was maintained throughout the time course of the experiments.

Statistical Analyses
Concentration–effect curves were analyzed by determining the maximal response and the concentration of agonist evoking 5% or 15% constriction (EC₅, EC₁₅). Statistical evaluation was performed using Student t test for either paired or unpaired observations. When >2 means were compared, analysis of variance was used: 1-way ANOVA with Dunnett post hoc test or 2-way ANOVA followed by Bonferroni post-hoc test (GraphPad Software, San Diego, Calif). Data are presented as mean±SEM, where n equals the number of animals from which blood vessels were taken or the number of cell culture experiments.

Reagents and Antibodies
The following reagents were used: fasudil hydrochloride (Tocris Cookson, Ellisville, Mo), H-1152 and Y27632 (Calbiochem, San Diego, Calif), Staphylococcus aureus -toxin (List Biological Laboratories, Campbell, Calif), fluo-4AM and Pluronic acid (Molecular Probes, Eugene, Ore), UK 14 304 (Sigma, St. Louis, MO), and MK912 (a gift from Merck, West Point, Pa). The following antibodies were used: mHA.11 antibody against the HA-epitope (Berkley Antibody, Berkley, Calif), mouse monoclonal RhoA antibody and mouse monoclonal ROCK-I antibody (BD Transduction Laboratories, Lexington, Ky), and an affinity-purified rabbit polyclonal antibody against the 2C-AR C-terminus (produced by Bethyl Laboratories, Houston, Tex).¹⁸ All secondary antibodies were purchased from Amersham. Silencing and nonsilencing RNA duplexes were obtained from Qiagen (Valencia, Calif).

An expanded Methods section can be found in the online data supplement available at http://circres.ahajournals.org.

	Results

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Rho Kinase Inhibitors and Arterial Constriction
Moderate cooling (to 28°C) increased the constriction evoked by the 2-AR agonist UK 14 304 in mouse tail arteries (Figures 1 and 2 ).⁷ Cooling caused a 4-fold leftward shift of the concentration–effect curve (log shift of 0.59±0.08, n=15, P<0.001, determined at 15% level of constriction) and increased the maximal response to the agonist by 78.1%+8.6% (n=15, P<0.001). Inhibition of Rho kinase by fasudil (3 µmol/L), Y27632 (0.3 µmol/L), or H-1152 (0.1 µmol/L)^23,24 did not affect the response to UK 14 304 at 37°C but significantly reduced constriction to the agonist at 28°C (Figures 1 and 2, and data not shown, respectively). At 28°C, the inhibitors caused rightward shifts in the concentration–effect curve of 7.3-fold (log shift of 0.86±0.23, n=5, P<0.05), 10.2-fold (log shift of 1.01+0.32, n=5, P<0.05), and 13.5-fold (log shift 1.13±0.31, n=5, P<0.05), respectively (determined at 15% level of constriction), and decreased the maximal response to the agonist by 38.4%±7.1%, 28.1%±6.4%, and 48.7%±9.5%, respectively (n=5, P0.01). In the presence of Rho kinase inhibitors, cooling no longer increased the constriction evoked by 2-AR stimulation with UK 14 304 (Figures 1 and 2).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of the Rho kinase inhibitor, fasudil (3 µmol/L, white symbols), on constriction evoked by the 2-AR agonist, UK 14 304 (0.3 to 100 nmol/L), in the mouse tail artery at 37°C (circles) or 28°C (squares) compared with control arteries in the absence of the inhibitor (black symbols). Vasoconstriction was expressed as a percentage of the baseline diameter and is presented as means±SEM for n=5. The symbols for the control curve at 37°C are hidden behind the fasudil (37°C) curve.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of the Rho kinase inhibitor, Y27632 (0.3 µmol/L, white symbols) on constriction evoked by the 2-AR agonist, UK 14 304 (0.3 to 100 nmol/L), in the mouse tail artery at 37°C (circles) or 28°C (squares) compared with control arteries in the absence of the inhibitor (black symbols). Vasoconstriction was expressed as a percentage of the baseline diameter and is presented as means±SEM for n=5.

Cold, 2-ARs, and Rho Activation
To directly assess the effects of cold and 2-AR stimulation on Rho activation, experiments were performed on VSMs cultured from human cutaneous arterioles, which express high endogenous levels of 2A-ARs and 2C-ARs.¹⁸ It was not possible to perform the assay on isolated arteries because of their small size.

Moderate cooling (28°C) caused a time-dependent activation of Rho observed over a time period of 5 to 60 minutes (Figure 3). The peak increase in Rho activation occurred after 30 minutes with a 63.1%±13.5% increase in activity (mean±SEM, n=4). In contrast, stimulation of 2-ARs by UK 14 304 (0.1 µmol/L) did not activate Rho at either warm (37°C) or cold (28°C) temperatures (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 3. Effect of cooling (to 28°C) on Rho activity in human cultured dermal arteriolar VSM cells. Cells were cooled to 28°C for increasing times and Rho activity determined by a pull-down assay, as described in the Methods. Active Rho was normalized to total Rho in the sample and expressed as a percentage increase in the basal activity at 37°C. Inset presents results of a typical experiment. The positive control was obtained by incubating the sample with GTPs (100 µmol/L). The bar graph presents the mean±SEM for 4 separate experiments. *Significant difference compared with control value (0 minutes), P<0.05.

Rho kinase-mediated inhibition of MLC phosphatase is associated with calcium sensitization of VSM contraction. Experiments were therefore performed in permeabilized arteries to assess functional activation of the Rho kinase pathway in mouse cutaneous arteries. Increasing the free calcium concentration from 1 nmol/L to 10µmol/L caused concentration-dependent constriction of permeabilized mouse tail arteries (Figure 4). Moderate cooling (to 28°C) significantly increased the sensitivity to calcium, causing a leftward shift in the concentration-response curve of 4.4-fold (log shift of 0.64±0.21, n=6, P<0.05, determined at 5% level of constriction), an effect that was reversible on warming. However, activation of 2-ARs by UK 14 304 (0.1 µmol/L) did not increase sensitivity to calcium at 37°C or 28°C (Figure 4). GTP (10 µmol/L) increased the sensitivity to calcium, causing a significant leftward shift in the curve (log EC₅ values of –5.94±0.10 and –6.36±0.15 in the absence and presence of GTP, respectively; n=6, P<0.05) (Figure 4). In the presence of GTP (10 µmol/L), neither UK 14 304 (0.1 µmol/L) nor cooling (to 28°C) affected the response to calcium (Figure 4).

View larger version (16K): [in this window] [in a new window]   Figure 4. Effect of increasing calcium concentration (0.01 to10 µmol/L) to cause constriction of -toxin–permeabilized mouse tail arteries, at 37°C (black symbols) and 28°C (white symbols). Sensitivity to calcium was assessed in the presence (triangles) and absence (squares) of UK 14 304 (0.1 µmol/L). Experiments were performed in the absence (upper panel) and presence (lower panel) of GTP (10 µmol/L). Vasoconstriction was expressed as a percentage of the baseline diameter (at 1 nmol/L calcium) and is presented as means±SEM for n=6.

Therefore, results from permeabilized arteries and cultured cells indicate that moderate cooling, but not 2-AR stimulation, causes activation of the Rho/Rho kinase signaling pathway.

Rho Kinase and Cold-Induced Translocation of 2C-ARs
Cold-induced augmentation of 2C-AR activity is highly selective for this contractile stimulus and is observed at early events in receptor signaling, including Gi protein-dependent inhibition of adenylyl cyclase.^2,7,8 This suggests that Rho kinase may augment 2C-AR responses by mechanisms in addition to increasing calcium sensitivity. A key event in cold-induced augmentation of 2C-AR activity is the translocation of the receptor from the Golgi compartment to the plasma membrane.⁸ To determine whether cold-induced translocation of 2C-ARs was mediated by Rho/Rho kinase signaling, experiments were performed in HEK 293 cells transiently transfected with HA-tagged 2C-ARs. The HA tag is at the N-terminus and would therefore be in the extracellular domain of cell surface receptors. By using a live-cell labeling technique directed against the HA tag, we confirmed that moderate cooling (to 28°C, 60 minutes) increased the localization of 2C-ARs to the cell surface (3.2±0.5-fold increase; mean±SEM; n=5, P<0.05; Figure 5) without affecting cellular expression of the receptor. Inhibition of Rho kinase by fasudil (10 µmol/L) prevented the cold-induced translocation of the 2C-ARs to the cell surface (Figure 5). Fasudil did not affect the low-level cell-surface expression of 2C-ARs present at 37°C or the total expression of the receptor.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effect of the Rho kinase inhibitor, fasudil, on the cold-induced mobilization of 2C-ARs to the plasma membrane in HEK293 cells. Experiments were performed in HEK 293 cells transfected with HA-tagged 2C-ARs. The HA tag is at the N-terminus and is therefore in the extracellular domain of cell surface receptors. The cells were maintained at 37°C, treated with or without fasudil (10 µmol/L) for 30 minutes, and then the temperature of the medium was changed to either 28°C or maintained at 37°C for 1 hour in the continued presence or absence of fasudil. The cells were then labeled with anti-HA antibody, and the cell-surface 2C-ARs subsequently immunoprecipitated from cell lysates with protein-A/G Sepharose beads. The inset images were obtained from a representative experiment and show the immunoprecipitated cell surface receptors, immunoblotted using anti-2C-AR antibody. The bar graph presents the mean±SEM for n=5. Moderate cooling caused a 3.2±0.5-fold increase in localization of 2C-ARs to the cell surface without affecting cellular expression of the receptor. Inhibition of Rho kinase by fasudil prevented cold-induced translocation of the 2C-AR receptor to the cell surface. a and b, Values with the same letter are significantly different, P<0.05.

To confirm a role for Rho kinase in the cold-induced translocation of 2C-ARs, RNA interference was used to decrease expression of ROCK-I in HEK 293 cells. Silencing RNA (siRNA) oligoduplexes targeted to ROCK-I decreased expression of the kinase by 71.0%±5.0% (n=6, P<0.001; Figure 6). Cotransfection of HEK 293 cells with 2C-ARs and a control nonsilencing RNA oligoduplex did not affect the cold-induced translocation of 2C-ARs to the cell surface, which increased by 5.2±1.2-fold (n=6, P<0.05; Figure 6). However, when expression of Rho kinase was reduced using siRNAs targeted to ROCK-I, cold-induced translocation of 2C-ARs was prevented (Figure 6). The low-level cell-surface expression of 2C-ARs present at 37°C and the total expression of the receptor were not affected by the siRNA duplexes. Therefore, Rho kinase mediates the cold-induced translocation of 2C-ARs to the plasma membrane.

View larger version (22K): [in this window] [in a new window]   Figure 6. Effect of Rho kinase inhibition, using RNA interference, on the cold-induced mobilization of 2C-ARs to the plasma membrane in HEK293 cells. Experiments were performed in HEK 293 cells cotransfected with HA-tagged 2C-ARs and control, nonsilencing RNA duplex (control or C) or siRNA duplexes targeted to ROCK-I. The upper inset demonstrates that siRNA (duplexes 1+4) reduced expression of ROCK-I (160-kDa band). Live cell labeling of the 2C-ARs at 37°C and 28°C was performed as described in Figure 5 and in Methods. The lower inset images were obtained from a representative experiment and show the immunoprecipitated cell-surface receptors, immunoblotted using anti-2C-AR antibody. The bar graph presents the mean±SEM for n=6. Moderate cooling caused a 5.2±1.2-fold increase in localization of 2C-ARs to the cell surface. Inhibition of Rho kinase by siRNA prevented cold-induced translocation of the 2C-AR receptor to the cell surface without affecting cellular expression of the receptor. a and b, Values with the same letter are significantly different, P<0.05.

Cold, Rho Kinase, and 2C-AR Signaling in Cutaneous Arteries
Cold-induced activation of Rho/Rho kinase could therefore contribute to enhanced 2C-AR constriction by 2 mechanisms: translocation of 2C-ARs to the plasma membrane and increased calcium sensitivity of the contractile process. To determine whether Rho/Rho kinase was involved in the functional rescue of the 2C-ARs in tail arteries, we analyzed an early signaling event initiated by 2-AR activation upstream of calcium sensitization of the contractile process, namely calcium mobilization. Isolated arteries were loaded with the calcium-sensitive fluorescent probe, Fluo4-AM, and VSM cells imaged using confocal laser microscopy. The 2-AR agonist UK 14 304 (0.1 µmol/L) caused calcium mobilization within VSMs at 37°C and 28°C (Figure 7). The selective 2C-AR antagonist, MK912 (0.3 nmol/L) did not significantly affect the response to UK 14 304 at 37°C but significantly reduced the response to the agonist at 28°C (Figure 7B). These results are consistent with the important role for 2C-ARs only during moderate cooling.⁷ Consistent with a role of Rho kinase in mediating the functional rescue of 2C-ARs, inhibition of Rho kinase by fasudil (3 µmol/L) reduced the response to UK 14 304 at 28°C, but not at 37°C (Figure 7C).

View larger version (35K): [in this window] [in a new window]   Figure 7. Effect of 2C-AR inhibition with MK912 (0.3 nmol/L) or Rho kinase inhibition with fasudil (3 µmol/L) on calcium mobilization caused by UK 14 304 (0.1 µmol/L) in mouse tail arteries. Isolated arteries were loaded with the calcium-sensitive fluorescent probe, Fluo4-AM, and fluorescence in arteriolar VSM cells imaged using confocal laser microscopy. A, The 2-AR agonist UK 14 304 (0.1 µmol/L) stimulated calcium mobilization within VSMs, and representative images are shown (left). The graph on the right presents the intracellular calcium concentration calculated from the fluo-4 fluorescence in arteries stimulated with UK 14 304 at 37°C (closed circles) or 28°C (open circles) (mean±SEM; n=13). B, The 2C-AR antagonist, MK912 (0.3 nmol/L; open circles) inhibited the increase in intracellular calcium in response to UK 14 304 in VSMs of tail arteries at 28°C (right panel; closed circles=control) but not at 37°C (left panel; closed circles=control). Fluorescence was expressed as a percent of baseline and is presented as means±SEM for n=4. C, Inhibition of Rho kinase by fasudil (3 µmol/L; open circles) reduced the response to UK 14 304 at 28°C (right panel; closed circles=control), but not at 37°C (left panel; closed circles=control). Fluorescence was expressed as a percent of baseline and is presented as means±SEM for n=4.

The aim of these experiments was primarily to examine the effects of Rho kinase inhibition on 2-AR function in the tail artery rather than to measure absolute changes in intracellular calcium concentration. However, Woodruff et al²⁵ determined the dissociation constant (K_D) of fluo-4 over a range of temperatures in mouse rod cells, which enables the estimation of calcium concentration in the present study. Maximum (Fmax) and minimum fluorescence (Fmin) values for fluo-4 were obtained after exposure of arteries to calcium ionophore, A23187 (10 µmol/L), or calcium-free Krebs solution containing EGTA (2 mmol/L), respectively. Intracellular calcium concentration in tail artery VSMs was then estimated according to [Ca2+]=K_D (F–Fmin)/(Fmax–F), where F is the fluorescence intensity.²⁵ At 37°C, UK 14 304 (0.1 µmol/L) increased the intracellular calcium concentration from an unstimulated level of 57.1±13.1 nmol/L to a peak level of 148.5±30.7 nmol/L. At 28°C, the effect of UK 14 304 (0.1 µmol/L) was significantly augmented, increasing calcium from an unstimulated level of 66.4±17.3 nmol/L to a peak level of 256.3±35.7 nmol/L (mean±SEM; n=13; P<0.01 for comparison of agonist responses at 37°C and 28°C; Figure 7A).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Cold-induced constriction of cutaneous blood vessels is mediated in part by a selective increase in the constrictor activity of VSM 2-ARs.^2–4 The mouse cutaneous artery model has demonstrated that 2-AR constriction is mediated by the 2A receptor subtype at 37°C, whereas after moderate cooling (28°C), the normally silent 2C-ARs mediate cold-induced augmentation of 2-AR responsiveness.⁷ Furthermore, unlike other 2-AR subtypes, the 2C-AR is largely associated with the Golgi compartment at 37°C and is translocated to the plasma membrane in response to cooling.⁸ The results of the present study demonstrate that the Rho/Rho kinase signaling pathway plays an important role in the cold-induced augmentation of 2-AR activity in cutaneous arteries. Cooling activates the Rho/Rho kinase pathway, which can then increase 2C-AR–mediated constriction by inducing translocation of 2C-AR to the plasma membrane and by increasing the calcium sensitivity of cutaneous VSM contraction.

Rho, a member of the Ras family of small GTP-binding proteins,^11–13 has been shown to be integral to many of the processes involving actin/myosin interactions in VSMs and other cells.^11,14 Inhibition of MLCP by Rho kinase brings about the shortening of actin/myosin filaments in a calcium-independent manner. This mechanism is considered to play a role in G protein-coupled receptor-mediated contraction in some vascular beds.^11,14,16 In the present study, inhibition of Rho kinase, by fasudil, Y27632, or H-1152 did not affect constriction to 2-AR stimulation at 37°C but reduced the 2-AR constriction at 28°C, abolishing the cold-induced augmentation in contractility. Therefore, Rho kinase plays an essential and selective role in the cold-induced modulation of 2-ARs. Indeed, experiments in human cultured cutaneous VSMs demonstrated that moderate cooling caused activation of Rho. Consistent with this observation, cooling increased the calcium sensitivity of contraction in permeabilized tail arteries. However, activation of 2-ARs, either at warm or cold temperatures, did not affect Rho activity in VSMs and did not alter the calcium sensitivity of permeabilized tail arteries. These results suggest that cooling, but not 2A or 2C-AR stimulation, is coupled to Rho/Rho kinase signaling. The mechanism by which moderate cooling activates Rho is not yet clear; however, one possibility is an interaction between microtubule structural dynamics and the release of guanidine nucleotide exchange factors that activate Rho.²⁶ Rho kinase has previously been implicated in constriction of rat arteries mediated by microtubule depolymerization.^27,28

The major mechanism by which the Rho/Rho kinase pathway has been linked to VSM contraction has been in causing calcium sensitization.^11,14 However, cold-induced augmentation of 2C-AR activity is highly selective for this stimulus, with constriction to other stimuli being inhibited during cold exposure.^2,7 Cold-induced alterations in calcium sensitivity would not be expected to impart such selectivity. Indeed, a key event in cold-induced augmentation of 2C-AR activity is the translocation of the receptor from the Golgi compartment to the plasma membrane.⁸ The Rho/Rho kinase pathway regulates the cytoskeletal structure of cells and is involved in regulating actin/myosin interactions, the formation of stress fibers, and the degree of polymerization of tubulin.^13,28 These processes are involved in trafficking membrane proteins between the endoplasmic reticulum/Golgi and the plasma membrane,^29,30 and Rho has been shown to play a role in the membrane targeting of the transmembrane protein, Na⁺K⁺-ATPase.¹⁷ To test the hypothesis that Rho activation played a role in the cold-induced translocation of 2C-ARs, an assay to selectively quantify the plasma membrane-associated receptor was used. This was performed using live cell labeling of HA epitope on the external amino terminus of the 2C-AR, transiently transfected into HEK293 cells. Immunofluorescence techniques demonstrated that the antibody did not gain access to intracellular sites. This technique was able to demonstrate a cold-induced increase in cell surface 2C-AR that was prevented by inhibition of Rho kinase using pharmacological (fasudil) or molecular targeting (RNA interference). This suggests that the Rho-Rho kinase pathway is involved in a novel pathway for regulating arterial constriction, namely the stimulated delivery of contractile receptors to the VSM cell surface.

We wished to confirm that this mechanism contributed functionally to cold-induced modulation of 2-AR activity in cutaneous arteries. Therefore, experiments were performed to analyze receptor signaling at a point upstream of MLC phosphorylation and VSM constriction, specifically the increase in VSM calcium mobilization in intact blood vessels in response to 2-AR stimulation. UK 14 304 caused a rapid increase in intracellular calcium in VSMs that preceded the onset of contraction. The potent and selective 2C-AR antagonist, MK912, at a concentration that does not inhibit 2A-ARs⁷ reduced the response to UK 14 304 during cold exposure, but not at 37°C. This finding is consistent with earlier observations, which demonstrated a switch in the functional activity of these receptors from 2A-ARs to 2C-ARs on cooling.⁷ Inhibition of Rho kinase with fasudil caused a marked reduction in the response to 2-AR stimulation in cold conditions while having no significant effect on the response at 37°C. This confirms that Rho kinase is not merely acting through calcium sensitization of the contractile process and plays an essential role to enable 2C-AR signaling at cold temperatures. Although the goal of these experiments was to examine modulation of the response rather than absolute changes in intracellular calcium concentration, estimation of those absolute changes demonstrated a significantly increased response to 2-AR stimulation at 28°C compared with 37°C.

Although 2C-ARs are important effectors in the cutaneous vascular response to cooling, they do not appear to respond directly to cold and therefore cannot be defined as "thermosensors." The temperature-sensitive mechanism responsible for mobilization of 2C-ARs appears to be a component of the Rho kinase signaling pathway. Further work is necessary to determine the factors responsible for cold-induced activation of this mechanism. Additional temperature-sensitive VSM mechanisms are likely to contribute to cold-induced vascular responses. For example, cooling causes prominent vasodilation in cutaneous blood vessels, which inhibits constriction to most stimuli.^2,4 Cold-induced dilation likely negates any generalized increase in constriction resulting from cold-induced increase in calcium sensitivity after activation of Rho/Rho kinase signaling. A notable exception to cold-induced depression of VSM contractility is the selective augmentation of 2-ARs.^2,4,7 The present study suggests this selectivity is mediated by cold-induced activation of the Rho/Rho kinase signaling pathway and the subsequent mobilization of 2C-ARs to the cell surface. Indeed, when 2C-ARs⁷ or Rho kinase (present study) is inhibited, cold actually decreases the 2-AR constrictor response.

In summary, the Rho/Rho kinase pathway plays a crucial role in the thermosensitivity of cutaneous arteries. Moderate cooling activates the Rho/Rho kinase pathway, which then can contribute to cold-induced vasoconstriction by stimulating the translocation of 2C-ARs to the cell surface and by increasing the calcium sensitivity of VSM contraction. Patients with Raynaud phenomenon and scleroderma exhibit increased vascular responsiveness to cold, which is mediated by 2-ARs.⁵ The present study suggests that Rho kinase may represent a novel therapeutic target for this disease process.

	Acknowledgments

 
This study was funded by grants from the National Institutes of Health (HL67331 and AR46126) and the Scleroderma Research Foundation.

	Footnotes

 
Received December 3, 2003; revision received April 1, 2004; accepted April 7, 2004.

	References

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